Naturally occurring interferons (IFNs) are species-specific proteins, often glycoproteins, produced by various cells upon induction with viruses, double stranded RNAs, other polynucleotides, antigens and mitogens. Interferons exhibit multiple biological activities such as antiviral, antiproliferative, immunomodulatory, and anticellular functions. At least three distinct types of human interferons have been identified and characterized in terms of their anti-viral, anti-growth and activation of natural killer cell (NK) activities. They are produced by leukocytes, lymphocytes, fibroblasts and the immune system and are classified as .alpha., .beta. and .gamma. interferons. These are reported to be different proteins coded for by distinct structural genes.
Native human .beta.-interferon (HuIFN-.beta.) is generally produced by superinducing human fibroblast cultures with poly-IC (polyriboinosinic acid and polyribocytidylic acid) and isolating and purifying the HuIFN-.beta. thus produced by chromatographic and electrophoretic techniques. Proteins or polypeptides which exhibit native .beta.-interferon activity may also be produced using recombinant DNA technology by extracting poly-A-rich 12S messenger RNA from virally induced human cells, synthesizing double-stranded c-DNA using the m-RNA as a template, introducing the c-DNA into an appropriate cloning vector, transforming suitable microorganisms with the vector, harvesting the bacteria and extracting the HIFN-.beta. therefrom. Nagola, S. et al., Nature, 284:316 (1980); Goeddel, D.V. et al., Nature, 287:411 (1980); Yelverton, E. et al., Nuc. Acid Res., 9:731 (1981); Streuli, M. et al., Proc. Nat'l. Acad. Sci. (U.S.), 78:2848 (1981); European Pat. Application Numbers 28033, published May 6, 1981; 321134, published Jul. 15, 1981; 34307 published Aug. 26, 1981; and Belgian Patent 837397, issued Jul. 1, 1981 describe various currently used methods for the production of .beta.-interferon employing recombinant DNA techniques. The expressed proteins or polypeptides have been purified and tested and have been found to exhibit properties similar to those of native IFNs. Bacterially produced IFNs thus appear to have potential therapeutic use as antiviral and anti-tumor agents and the production of IFNs by such bacterial fermentations is expected to yield sufficiently large quantities of IFN at a relatively low cost for clinical testing.
Further, HuIFN-.beta. genes have been altered by, for example, oligonucleotide-directed mutagenesis to produce IFN-.beta. protein analogs thereof, such as the human recombinant cysteine-depleted or cysteine-replaced interferon-B analogs (muteins) disclosed in U.S. Pat. No. 4,588,585 issued May 13, 1986 to Mark et al. Specifically disclosed in that patent is the recombinant IFN-.beta. mutein wherein the cysteine at latter IFN-.beta. mutein is IFN-.beta..sub.ser17.
Procedures for recovering and purifying bacterially produced IFNs are described in U.S. Pat. Nos. 4,450,103; 4,315,852; 4,343,735; and 4,343,736; and Derynck et al., Nature (1980) 287:193-197 and Scandella and Kornberg, Biochemistry. 10:4447 (1971). Generally with these methods the IFN is not produced in a sufficiently pure form and in sufficiently large quantities for clinical and therapeutic purposes and the resulting IFN preparations produced by recombinant DNA techniques have residual amounts of chemicals, such as sodium dodecyl sulfate (SDS) and other surfactants or precipitants used in the extraction and purification steps.
E. coli expressed recombinant IFN-.beta. and analogs thereof are insoluble in solutions which are at a pH range of 6 to 9. Therefore, various processes and additives have been devised to solubilize these proteins. Several methods currently available for the preparation, recovery and purification of bacterially produced proteins are listed immediately below.
U.S. Pat. No. 4,315,852 to Leibowitz et al. describes a method for the acid extraction of leukocyte interferon from bacterial cells and neutralization of the extractant to obtain the interferon.
U.S. Pat. No. 4,343,735 to Menge et al. teaches a process for the purification of interferon by partitioning it in an aqueous multi-phase system in the presence of ion exchangers which are soluble in the system and are derivatives of polyethers.
U.S. Pat. No. 4,343,736 to Uemura et al. discloses a method for recovering interferon by absorption of water-insolubilized heparin and then eluting the interferon with an aqueous solution of an inorganic salt and chondroitin sulfate.
U.S. Pat. No. 4,289,689 to Friesen et al. discloses how to recover and purify human native .beta.-interferon by use of affinity chromatography and high pressure liquid chromatography.
U.S. Pat. No. 4,364,863 to Leibowitz et al. describes a method of extracting fibroblast interferon from bacteria using a low pH followed by a high pH extraction procedure.
U.S. Pat. No. 4,450,103 to Konrad et al. discloses solubilizing the protein in an aqueous medium with an appropriate solubilizing agent, extracting the protein from the aqueous medium with 2-butanol or 2-methyl-2-butanol, and precipitating the protein from the alcohol phase.
U.S. Pat. No. 4,530,787 to Shaked et al. describes a process for oxidizing recombinant proteins such as IFN-.beta. selectively and stoichiometrically using o-iodosobenzoic acid to ensure that the protein will be functionally equivalent to its native counterpart.
Many heterologous proteins are precipitated intracellularly in the form of refractile or inclusion bodies which appear as bright spots visible within the enclosure of the cell under a phase contrast microscope at magnifications down to 1000 fold. See e.g., Miller et al., Science (1982) 215:687-690; Cheng, Biochem. Biophys. Res. Comm., (1983) 111:104-111; Becker et al., Biotech. Advs. (1983) 1:247-261; Kleid et al., ch. 25 in Developments in Industrial Microbiology, Vol. 25, p. 317-325 (Society for Industrial Microbiology, Arlington, Va., 1984); Marston et al., Bio/Technology. (September, 1984), pp. 800-804.
Purification and activity assurance of precipitated heterologous proteins is also described by U.S. Pat. Nos. 4,511,502; 4,511,503; 4,512,922; 4,599,127 and 4,518,526; and EP 114,506.
Commonly owned U.S. application Ser. Nos. 749,951 filed Jun. 26, 1985 now abandoned and U.S. Ser. No. 843,997, filed Mar. 25, 1986, now U.S. Pat. No. 4,748,234, both to Dorin et al., and entitled "Process for Recovering Refractile Bodies Containing Heterologous Proteins from Microbial Hosts" disclose improved methods for recovering and purifying refractile bodies. To isolate the refractile material, the processes initially involve disrupting the cell wall and membrane of the host cell, removing greater than 99% by weight of the salts from the disruptate, redisrupting the desalted disruptate, adding a material to the disruptate to create a density or viscosity gradient in the liquid within the disruptate, and separating the refractile material from the cellular debris by high-speed centrifugation. The heterologous protein, preferably recombinant IFN-.beta. or interleukin-2 (IL-2), is then extracted from the refractile bodies and solubilized with a denaturing agent such as sodium dodecyl sulfate (SDS). The SDS is later removed by a desalting column.
Wang et al., J. Parenteral. Drug Assoc; 34, 452-462 (1980) provides a review of excipients and pHs for parenteral products used in the United States. A list of solubilizing agents such as detergents and lipids in use for various drugs is provided in Table I thereof and under section 11 entitled "Solubilizers, Wetting Agents or Emulsifiers" of that table, polyethylene glycol 300, polysorbate 20, 40 and 80, and propylene glycol among others are listed for a variety of pharmaceuticals. Below are referenced some examples of interferon and related formulations.
U.S. Pat. No. 4,647,454 to Cymbalista et al. discloses a method of stabilizing human fibroblast interferon with polyvinyl pyrrolidone.
U.S. Pat. No. 4,460,574 to Yabrov discloses a pharmaceutical composition comprising native human .alpha.- and .beta.-interferons used for rectal or urogenital treatment of human interferon-sensitive diseases.
U.S. Pat. No. 4,462,940 to Hanisch et al. ( '940 patent) discloses a process for formulating interferon by mixing the interferon and a protein stabilizer, such as normal serum albumin, at a pH of about 10.5 to 12.5 for 5 minutes and then adjusting the pH to 7.5 to obtain a soluble mixture. The '940 patent to Hanisch et al. further describes a method for recovering and purifying a lipophilic protein such as human recombinant interferon-.beta. or IL-2 which comprises solubilizing the protein into an aqueous medium with a suitable solubilizing agent, such as sodium dodecyl sulfate (SDS), extracting the solubilized protein with an aliphatic alcohol, precipitating the protein from the alcohol phase with an aqueous buffer, and diafiltering the protein at a pH of about 10.5 to 12.5 against water or against mixtures of water and aliphatic alcohols adjusted to a pH of about 10.5 to 12.5, to remove the solubilizing agent (SDS) or reduce its concentration.
One aspect of the instant invention concerns a process alternative to the method described above and claimed in U.S. Pat. No. 4,462,940. Said process alternative avoids the high pH range (pH 10.5 to 12.5) required to remove certain solubilizing agents such as SDS during diafiltration or desalting, preferably desalting.
Commonly owned, U.S. application Ser. No. 775,751, filed Sept. 13, 1985 now U.S. Pat. No. 4,992,271, entitled "An Improved Formulation for Lipophilic Proteins" outlines a high pH and a low pH process for recovering and purifying lipophilic recombinant proteins, such as human IFN-.beta. and interleukin-2, from host strains to yield a protein preparation which may be formulated into a stable pharmaceutical composition. Said composition carrying a therapeutically effective amount of the biologically active lipophilic protein dissolved in a non-toxic, inert, therapeutically compatible aqueous-based carrier medium at a pH of 6.8 to 7.8 also contains a stabilizer for the protein, such as human serum albumin, human serum albumin and dextrose, or human plasma protein fraction.
Commonly owned, U.S. application Ser. No. 780,551, filed Sept. 26, 1985, now U.S. Pat. No. 4,816,440, entitled "Stable Formulation of Biologically Active Proteins for Parenteral Injection," discloses pharmaceutical compositions containing IFN-.beta. or interleukin-2 dissolved in a stable carrier medium at pH 7.0 to 8.0 stabilized with sodium laurate.
Commonly owned, U.S. application Ser. Nos. 749,955. filed Jun 26, 1985, now abandoned and Ser. No. 866,459 filed May 21, 1986 (Katre et al.), now U.S. Pat. No. 4,766,106, disclose pharmaceutical compositions wherein recombinant IFN-.beta., IL-2 or an immunotoxin is dissolved in an aqueous carrier medium at pH 6 to 8 without the presence of a solubilizing agent. The protein is solubilized by selectively conjugating it via a coupling agent to a water-soluble polymer selected from polyethylene glycol homopolymers or polyoxyethylated polyols.
Japanese Laid-Open Patent Application (Kokai) No. 59-10524 (published Jan. 20, 1984) entitled "An Interferon Composition and a Method of Manufacturing the Same" discloses a micelle solution for rectal administration prepared by mixing (a) an unsaturated fatty acid, (b) a polyoxyethylene fatty acid ester, alkyl polyoxyethylene ether or sucrose fatty acid ester, (c) water and (d) interferon.
European Patent Application Publication No. 135,171 (published Mar. 27, 1985) discloses pseudomonophase, microemulsion compositions for topical application of interferons, preferably leukocyte interferon A. The compositions comprise a therapeutically effective amount of interferon, 30-70% by volume of a surface active agent having a hydrophilic-lipophilic balance (HLB) of from 12-15 and dual solubility in water/oil; 5-45% of a vegetable oil; and 5-45% water. The surface active agents disclosed therein are polyethylene glycol derivatives of castor oil composed on average of 25-36 moles of ethylene oxide per mole of castor oil. Such an oil-based microemulsion is not stable and is subject to phase separation.
U.S. Pat. No. 4,507,281 discloses compositions comprising about 10.sup.4 to 10.sup.6 I.U. of human leukocyte interferon, about 1% to 5% by weight of a non-ionic surface active agent having at least one ether or amide linkage, and a physiologically acceptable carrier. Such compositions are topically administered to treat herpes simplex infections wherein a therapeutic effect is attributed to such non-ionic surfactants in their ability to dissolve the lipid-containing envelope of the herpes simplex virus. The preferred non-ionic surfactants referred to therein include: nonylphenoxypolyethoxy ethanol (trade name Nonoxynol-9); p-diisobutylphenoxypolyethoxy ethanol (tradename Triton X-100); polyoxyethylene (10) oleyl ether (trade name Brij-97); and onyx-ol (trade name Onyx-ol 345). Such pharmaceutical compositions are preferably administered in lotion, cream, oil, or emulsion formulations. See also European Patent Application Publication No. 77,063 which is a foreign equivalent thereof.
There remains a need in the art for formulations of biologically active, recombinant beta-interferons that are pure enough for clinical administration but substantially or totally free of residual strong detergents, such as SDS, used in the extraction and purification processes. Further, there is a need for formulations that provide alternatives to those containing non-IFN-.beta. protein, such as those containing albumin.
Further, alternative formulation processes for such biologically active, recombinant beta-interferons which avoid very high pH ranges are desirable.